A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences

Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is an essential tool in understanding and affecting their roles. Adeno-associated viruses (AAVs) enable rapid genetic manipulation; however, astrocyte specificity of AAVs can be limited, with high off-target expression in neurons and sparsely in endothelial cells. Here, we report the development of a cassette of four copies of six miRNA targeting sequences (4x6T) which triggers transgene degradation specifically in neurons and endothelial cells. In combination with the GfaABC1D promoter, 4x6T increases astrocytic specificity of Cre with a viral reporter from <50% to >99% in multiple serotypes in mice, and confers astrocyte specificity in multiple recombinases and reporters. We also present empty vectors to add 4x6T to other cargo, independently and in Cre/Dre-dependent forms. This toolbox of AAVs allows rapid manipulation of astrocytes throughout the CNS, is compatible with different AAV serotypes, and demonstrates the efficacy of using multiplexed miRNA targeting sequences to decrease expression in multiple off-target cell populations simultaneously.


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Experiments were independently performed at least 3 times, with the exception of negative control inducible Cre experiments, which were replicated twice (no tamoxifen for ER-Cre-ER induction, no light for iCreV induction).All replicates for each experiment had similar results.
For transcriptomic experiments, male and female Ribotag+ mice 2-5 months were distributed across viral groups such that each group included both sexes and animals across the age range.For immunohistochemistry experiments, animals were distributed across viral groups such that each group included both sexes and animals from multiple cages.
Identical protocols were used to process and analyze all samples.For direct comparison of the efficacy of different individual miR targeting sequences and cell-type specificity in different serotypes, experimenters were blinded during analysis.The nature of other comparisons prevented blinding: comparing different brain regions, systemic vs targeted viral expression, different reporters with different visual appearance, and comparing animals of different ages (due to the heavy presence of lipofuscin in 28m animals).
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